The NMDA receptor is a major mediator of the neuronal plasticity that occurs in the spinal cord dorsal horn (SCDH) and its activation results in the facilitation of injury-induced pain and to the development of opioid tolerance. Following CFA-induced inflammation, a spatial functional knockout (KO) of the NMDA receptor (NR1) that we have developed, prevents evoked allodynia. This protective effect is lost with time suggesting the onset of a transcriptionally mediated NMDA receptor-independent pain process. In this revised application we will use the spatial NR1 KO mouse to identify the contribution of selected spinal cord dorsal horn (SCDH) neuronal and glial proteins (dynorphin, NK-1 receptors, COX-2, ERKI/2, p38 MAPK and proinflammatory cytokines) to activated pain states in the presence and absence of NMDA receptors (Aim 1). The expression levels of these proteins will be measured (immunocytochemistry, Western blot, in situ hybridization, RT PCR and ribonuclease protection assays) in SCDH after inflammation or nerve injury to determine which proteins are upregulated and whether this increased expression is NMDA receptor dependent. Inhibitors of these proteins will be evaluated for their efficacy in reducing activated pain states (evoked allodynia) in these animals. The NR1 KO mouse will also be used to define the contribution of SCDH NMDA receptors to systemic and intrathecal morphine tolerance. Recombinant adenoassociated viral (rAAV) vectors will be used to deliver therapeutic molecules in the form of RNAi, antisense or anti- inflammatory cytokines in a spatially directed manner to the SCDH so as to validate the in vivo utility of targeting selected pain activated proteins (Aim 2). A strategy is presented to design and test shRNAs that will be processed in vivo into siRNAs that will cleave our proof of principle target, NR1 mRNA resulting in a spatial knockdown of NR1. A complementary approach will use a rAAV vector to spatially localize the expression of anti-inflammatory cytokines in SCDH. In vitro and in vivo studies will be used to evaluate the properties of these vectors. The proposed research will improve our understanding of the contribution of the NMDA receptor to the pain activated protein cascade, identify therapeutic targets and will provide new gene expression based therapeutic approaches for the management of pain and opioid tolerance.